The pathogenesis of asthma and allergy is marked by dysregulated expression of the Th2 cytokines IL4 (which is critical for Th2 differentiation and IgE synthesis) and IL13 (which is the central effector of allergic inflammation). We seek to dissect the regulation of human Th2 cytokine genes and define the mechanisms by which natural polymorphisms affect their expression and/or function, thereby influencing allergy susceptibility. During the previous funding cycle, we investigated the chromatin-based mechanisms that regulate IL13 expression in human CD4 T cells. We identified novel elements (HS4, HS5, HS11/12) marked by the convergence of three indicators of cis-regulatory function: DNase I hypersensitivity, DNA hypomethylation and sequence conservation. In parallel, we showed that IL13 single nucleotide polymorphisms (SNPs) associated with increased susceptibility to allergy/asthma, and located in HS4 and HS5, increase IL13 transcription in vitro. To advance the field, we then developed a powerful in vivo model to explore both human Th2 cytokine regulation and its modulation by genetic variants. This model relies on mice carrying a 160 kb BAC transgene (BAC5) encompassing human RAD50, IL13 and IL4. Human IL13 and IL4 are faithfully regulated in BAC5 TG murine T cells, suggesting BAC5 contains all the cis-acting elements that control human Th2 cytokines. With support from a separate grant (R21A1076715, PI: Vercelli), we are studying the impact of natural variants on human IL13 expression using BAC5 TG mice that carry wild-type or allergy-associated human IL13 alleles. Unexpectedly, mice with SNPs in HS4, HS5 and HS11/12 exhibited not only a significant, albeit modest, increase in human IL13, but also striking (3-fold) increases in human IL4. Notably, children enrolled in the Infant Immune Study who carry the same SNPs showed comparable Th2 cytokine patterns. These data suggest that HS4, HS5 and/or HS11/12 contain novel cis-element(s) primarily involved in human IL4 control. This competing renewal application builds on our chromatin analyses and our new TG model and proposes to characterize these novel IL13/IL4 cis-regulatory regions in vivo. We will generate BAC5 TG mice carrying Cre-mediated deletions of HS4, HS5 or HS11/12, and we will assess the impact of these deletions on human Th2 cytokine expression, Th2 locus chromatin architecture and long-range intra-chromosomal interactions. We will focus on IL4 as well as IL13 because significant effects on IL4 are likely, and would have major implications for the pathogenesis of allergic disease. We will examine CD4 Th cells at distinct differentiation stages and, albeit less extensively, mast cells. Specific objectives are to define the role of HS4 (Aim 1) and HS5 (Aim 2), which reside in the distal IL13 promoter and are the only IL13 HS sites detected in naive human CD4 T cells;and to characterize HS11/12, which lies in the IL13/IL4 intergenic region and is the most intense IL13 HS site (Aim 3). Our studies will be the first to explore human Th2 cytokine regulation in vivo and will identify novel rational targets for effective treatments of allergic disease.